An influenza A virus nucleoprotein (NP) gene was cloned from DNA sequences derived by reverse-transcription of virion RNA segments using synthetic oligonucleotide primers as detailed previously. The cloned NP DNA coding for a full-length copy of a virion NP RNA segment was verified by the size and the completeness of DNA sequences at both terminal ends. The influenza A virus nucleoprotein (NP) gene was cloned into the BamH1 site of the late region of SV40 in an SV40-pBR322 expression vector. African green monkey kidney primary cells transfected with the SV40-NP recombinant DNA in the presence of an early SV40 ts mutant helper, synthesized a polypeptide that was specifically immunoprecipitable with NP monoclonal antibodies and that had a molecular weight of 56 K daltons identical to the NP of influenza virus as estimated on SDS-polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an indirect immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection suggesting the NP product may be functionally active.